L-Theanine Activates the Browning of White AdiposeTissue Through the AMPK/a-Ketoglutarate/Prdm16 Axisand Ameliorates Diet-Induced Obesity in MiceWan-Qiu Peng,1 Gang Xiao,1 Bai-Yu Li,1 Ying-Ying Guo,1 Liang Guo,1,2 and Qi-Qun Tang1

Diabetes 2021;70:1458–1472 | https://doi.org/10.2337/db20-1210

L-Theanine is a nonprotein amino acid with much bene-ficial efficacy. We found that intraperitoneal treatmentof the mice with L-theanine (100 mg/kg/day) enhancedadaptive thermogenesis and induced the browning ofinguinal white adipose tissue (iWAT) with elevatedexpression of Prdm16, Ucp1, and other thermogenicgenes. Meanwhile, administration of the mice with L-theanine increased energy expenditure. In vitro studiesindicated that L-theanine induced the development ofbrown-like features in adipocytes. The shRNA-mediateddepletion of Prdm16 blunted the role of L-theanine in pro-moting the brown-like phenotypes in adipocytes and inthe iWAT of mice. L-theanine treatment enhanced AMPKaphosphorylation both in adipocytes and iWAT. Knock-down of AMPKa abolished L-theanine–induced upregula-tion of Prdm16 and adipocyte browning. L-Theanineincreased the a-ketoglutarate (a-KG) level in adipocytes,which may increase the transcription of Prdm16 by induc-ing active DNA demethylation on its promoter. AMPK ac-tivation was required for L-theanine–induced increase ofa-KG and DNA demethylation on the Prdm16 promoter.Moreover, intraperitoneal administration with L-theanineameliorated obesity, improved glucose tolerance and in-sulin sensitivity, and reduced plasma triglyceride, totalcholesterol, and free fatty acids in the high-fat diet–fedmice. Our results suggest a potential role of L-theanine incombating diet-induced obesity in mice, which may in-volve L-theanine–induced browning of WAT.

Obesity has become a global epidemic and is a major riskfactor associated with several metabolic syndromes, suchas type 2 diabetes, insulin resistance, heart disease,stroke, hyperglycemia, hypertension, and cancer (1,2). Ad-ipose tissue is a key metabolic organ. Adipose tissue com-position is always in a dynamic process of change.Adipocytes play critical roles in whole-body energy metab-olism and homeostasis, which can be classified into threetypes. White adipocytes store excess energy in lipid drop-lets (3), while beige and classical brown adipocytes arecharacterized by their unique ability to transform mito-chondrial energy into heat via uncoupling protein 1(Ucp1) (4).

Mounting evidence suggests that browning of whiteadipose tissue (WAT) is of great benefit to human meta-bolic health. It can not only burn fat, but also enhance theinsulin sensitivity of the body and reduce the fibrosisand inflammatory response of WAT, which plays an impor-tant role in maintaining the metabolic homeostasis. Com-pound supplementation that promotes browning of whiteadipocytes provides a therapeutic option to ameliorateobesity.

Natural products have been used for medical purposesfor a long time. Many kinds of teas are consumed widelyfor health care needs. Growing studies suggest that drink-ing green tea reduces the risk of obesity and related disor-ders (5). Green tea is enriched with many secondary

1Key Laboratory of Metabolism and Molecular Medicine of the Ministry ofEducation, Department of Biochemistry and Molecular Biology of School of BasicMedical Sciences and Department of Endocrinology and Metabolism ofZhongshan Hospital, Fudan University, Shanghai, China2School of Kinesiology, Shanghai University of Sport, Shanghai, China

Corresponding authors: Qi-Qun Tang, qqtang@shmu.edu.cn, and Liang Guo,guoliang@sus.edu.cn

Received 30 November 2020 and accepted 12 April 2021

This article contains supplementary material online at https://doi.org/10.2337/figshare.14414216

© 2021 by the American Diabetes Association. Readers may use this articleas long as the work is properly cited, the use is educational and not forprofit, and the work is not altered. More information is available at https://www.diabetesjournals.org/content/license.

METABOLISM

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metabolites, such as catechins, caffeine, and L-theanine(6). For a long time, attention was significantly focusedon catechins and caffeine for their metabolic benefits.However, in recent years, there is increasing attention onthe health benefits of L-theanine. L-Theanine accounts for1–3% of the dry weight of tea, which varies according togrowing environment, variety, collection, and productionprocess of the tea (7). L-Theanine was reported to im-prove sleep quality, emotional state, and cognitive perfor-mance and to combat cancer, cardiovascular diseases, andthe common cold (8). However, its role in adipose tissuemetabolism and antiobesity effects are poorly understood.

In this study, we showed that an L-theanine interven-tion significantly induced the browning of subcutaneousWAT in mice. The molecular mechanism underlying theabove role of L-theanine was dissected both in vitro andin vivo. We have demonstrated that L-theanine could reg-ulate a thermogenic program in WAT, and these effectsare AMPK/a-ketoglutarate (a-KG)/Prdm16 axis–depend-ent. Our data clarified a previously unknown role of L-the-anine in increasing energy expenditure, which might havea prospective application in improving metabolism andcombating obesity-related metabolic diseases.

RESEARCH DESIGN AND METHODS

Cell Culture and ReagentsC3H10T1/2 cells were obtained from the ATCC (Manassas,VA). For the ex vivo system, iWAT from wild-type C57BL/6mice was fractionated with digestion buffer. Primary iWATstromal vascular fractions (SVFs) were fractionated by collage-nase digestion and plated in culture according to the methodsdescribed previously (9). C3H10T1/2 cells and primary SVFswere cultured in complete DMEMwith 10% FCS at 37 degreesand 5% CO2. Adipocytes were differentiated with an adipogeniccocktail (0.5 mmol/L isobutylmethylxanthine, 1 mmol/L rosigli-tazone, 1mg/mL insulin, and 1mmol/L dexamethasone) in me-dium containing 10% (v/v) FBS (Invitrogen). After 2 days, themedium was exchanged for DMEM supplemented with 10%(v/v) FBS, 1 mg/mL insulin, and 1 mmol/L rosiglitazone foranother 2 days. Media was changed every other day startingfrom the 4th day postdifferentiation with DMEM containing10% (v/v) FBS. Cells were treated with L-theanine and infectedwith adenoviruses from the 6th day, and cell cultures were har-vested 48 h later. Insulin, dexamethasone, and isobutylmethyl-xanthine were purchased from Sigma-Aldrich. Rosiglitazone waspurchased from Cayman Chemical. The primary antibodiesused in this study are as follows: anti-Ucp1 (ab209483; Abcam);anti-PGC1a (ab54481; Abcam); anti-Prdm16 (ab106410; Ab-cam); anti-AMPK (detecting AMPKa1, 5832; Cell Signal-ing Technology); anti–phospho-AMPK (Thr-172 ofAMPK, 2535; Cell Signaling Technology); HSP-90 (4874;Cell Signaling Technology); 5-hydroxymethylcytosine (5-hmc)(A4001; Zymo Research); 5-methylcytosine antibody (5-mc)(A3001; Zymo Research); and anti–b-actin (sc-47778; SantaCruz Biotechnology). L-Theanine was purchased from

Shandong Topscience Biotech Co., Ltd. (T2800) and dissolvedin saline. Hoechst was from US Everbright Inc. (H4047), andMitoTracker Green (MTG) was from Beyotime Biotechnology(C1048). Oil Red O stock solution (0.25 g of Oil Red O[Sigma-Aldrich] in 50 mL of isopropanol) was diluted with wa-ter (3:2 v/v), followed by filtration. After staining, sampleswere washed three times in water.

AnimalsOur animal protocol has been reviewed and approved by theAnimal Care and Use Committee of the Fudan UniversityShanghai Medical College (20180302–010). Mice were main-tained under a 12:12-h light/dark cycle at room temperature(25�C) with free access to food and water. For the cold tolerancetest, mice were placed in a temperature-controlled chamber(Friocell; MMMGroup,M€unchen, Germany). Body temperaturewas measured using an animal electronic thermometer (ALT-ET03; Shanghai Alcott Biotech Co. Ltd.). For high-fat feedingstudies, male C57BL/6J mice were put on the diet beginning at6 weeks of age and continuing for up to 16 weeks. Blood andvarious tissue samples were collected. Animals were given L-the-anine or saline by intraperitoneal (i.p.) injection for 7 days atroom temperature during chow diet or for 10 consecutiveweeks during high-fat diet (HFD). For metabolic studies, micewere housed and monitored individually in metabolic cages (Co-lumbia Instruments International) with free access to regularchow and drinking water for 48 h. The animals were acclimatedto the system for 24 h, and measurement of VO2 and VCO2

was performed during the next 24 h. Each cage was monitoredfor metabolic parameters (including oxygen consumption andcarbon dioxide production) at 25-min intervals throughout the48-h period. Parameters of oxygen consumption (in millilitersper kilogram per hour), carbon dioxide production (in millilitersper kilogram per hour), heat (in kilocalories per hour), and re-spiratory exchange ratio (RER) (as VCO2/VO2) were calculatedfor each mouse divided by its body weight. Body fat and leanmass were measured using an NMR analyzer (Minispec LF90II;Bruker Optics). The serum total triglyceride (TG) and total cho-lesterol (TC) were measured using a Fully Automatic Biochemi-cal Analyzer (Roche) according to the manufacturer’s protocol.The plasma free fatty acid (FFA) levels of the mice were mea-sured by using the FFA assay kit from Abcam.

Glucose Tolerance Test and Insulin Tolerance TestFor the glucose tolerance test (GTT), mice were subjected tofasting at 6:00 P.M. On the next day at 10:00 A.M., GTT wasconducted through i.p. injection of glucose at 2 g/kg bodyweight. For the insulin tolerance test (ITT), mice were sub-jected to fasting for 5 h from 7:00 A.M. to 12:00 P.M., afterwhich ITT was performed by i.p. injection of human insulin(0.5 units/kg body weight). Glucose level was measured intail blood at the indicated time after glucose or insulin ad-ministration using a glucometer (Accu-Chek; Roche).

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Hematoxylin and Eosin Staining andImmunohistochemistryFor histology, adipose tissues were fixed with neutral-buffered formalin and embedded in paraffin, and sectionswere stained with hematoxylin and eosin (H&E). Immu-nohistochemistry (IHC) was performed as described previ-ously (10). The anti-Ucp1 (ab209483; Abcam) antibodywas used for IHC. Images were captured using a charge-coupled device camera, and representative images areshown.

RNA Extraction and Quantitative PCRTotal RNA from adipose and adipocyte samples was iso-lated using TRIzol (Invitrogen) reagent according to themanufacturer’s instructions and reverse-transcribed withSuperScript III Reverse Transcriptase (Invitrogen). cDNAwas reverse-transcribed from 1 mg of RNA. QuantitativePCR (QPCR) was performed with SYBR Green QPCR Mas-ter Mix (Applied Biosystems, Carlsbad, CA) using a Prism7500 instrument (Applied Biosystems), with 18S rRNA or36b4 as an endogenous control. All primer sequences arelisted in Supplementary Table 1. Expression levels werecalculated according to the relative 2�DDCt method (11).

Western Blotting AnalysisWhole-cell protein lysates were prepared using lysis buffercontaining 1% SDS, 50 mmol/L Tris-HCl (pH 6.8), 10mmol/L dithiothreitol, 10% glycerol, 0.002% bromophe-nol blue, 1 mmol/L sodium fluoride (Sigma-Aldrich), 1mmol/L sodium orthovanadate (Sigma-Aldrich), and pro-tease inhibitor cocktail (Complete Mini EDTA-free; RocheApplied Science). Protein was resolved using Tris-glycinegels and transferred to polyvinylidene difluoride mem-brane (Millipore, Bedford, MA). After blocking with 5%nonfat dried milk in Tris-buffered saline with Tween, themembranes were incubated with the primary antibodiesand secondary antibodies and visualized with horseradish per-oxidase-coupled secondary antibodies.

Oxygen Consumption AssaysCultured adipocytes were detached from plates by trypsin.Cells or minced tissues were then resuspended in 1 mLDulbecco’s PBS (25 mmol/L glucose, 1 mmol/L pyruvate,and 2% BSA). Cellular respiration was measured with aClark-type oxygen electrode (Oxygraph1 system; Hansa-tech Instruments Ltd.). Data were normalized by totalprotein content or tissue weight.

MTG StainingCultured cells were incubated in DMEM with MTG (finalconcentration 200 nmol/L; C1048; Beyotime Biotechnology)for 20 min in a 37�C incubator and then washed two timesin PBS. The cells were observed and photographed under anAxioskop 2 microscope (Carl Zeiss) with a DP70 charged-cou-pled device system (Olympus). Cell nuclei were labeled withHoechst.

Lactate Dehydrogenase ActivityCytotoxicity was determined in a colorimetric assay basedon the measurement of lactate dehydrogenase (LDH) re-leased from the C3H10T1/2 cells into the supernatant ac-cording to the method (12). The LDH release activity wasdetermined using an LDH cytotoxicity assay kit (BeyotimeBiotechnology) according to the manufacturer’sinstructions.

Analysis of mtDNA Content by Quantitative Real-timePCRCells and tissues were homogenized and digested with protein-ase K in a lysis buffer for DNA extraction by using the DNeasyBlood and Tissue Kit (Qiagen). The ratio of mtDNA to nuclearDNA reflects the concentration of mitochondria per cell (13).The Prism 7500 instrument (Applied Biosystems) was used toamplify nuclear DNA (forward, 50-GCCAGCCTCTCCTGATTT-TAGTGT-30 and reverse,50-GGGAACACAAAAGACCTCTTCTG-G-30) and mtDNA (forward, 50-CCGCAAGGGAAAGATGAAAGA-30 and reverse, 50-TCGTTTGGTTTCGGGGTTTC-30). The resultswere calculated from the difference in the threshold cycle (DCT)values for mtDNA and nuclear-specific genes.

Construction of Adenoviral Expression Vectors andInfectionAdenoviral expression vector pBLOCK-iT (Invitrogen) en-coding shRNA against LacZ, Prdm16, and AMPKa wasconstructed according to the manufacturer’s protocol. Thetargets of shRNA were listed as: shRNA against LacZ(shLacZ), 50-CTACACAAATCAGCGATTT-30; shRNA againstPrdm16 (shPrdm16), 50-AGTGACTTTGAGGATATCA-30;shRNA against AMPKa1 (shAMPKa1), 50-GCACACCCTG-GATGAATTAAA-30; and shRNA against AMPKa2 (shAM-PKa2), 50-GCTGAGAACCACTCCCTTTCT-30. Adenovirus vectorswere amplified and purifiedwith Sartorius Adenovirus Purificationkits. Viral titers were determined by the 50% tissue culture infec-tious dose method using 293A cells. For in vivo studies, 109

plaque-forming units/mouse of adenoviruses harboring shLacZ orshPrdm16 were diluted to 200mL with PBS, and then 100mL ofadenovirus solution was injected to the subcutaneous fat pads ateach side, twice a week as indicated. For in vitro studies, adenovi-ruses harboring shLacZ, shPrdm16, or shAMPKa at a multiplicityof infection of 70 were added to the cell culture medium. Then,cells were cultured with the viruses for 48 h and then replacedwith fresh culture medium. Experiments were performed 48 h af-ter adenovirus infection.

a-KG AssayThe a-KG contents in iWAT and the adipocytes derivedfrom C3H10T1/2 cells were analyzed using liquid chroma-tography-tandem mass spectrometry. C3H10T1/2 cellswere cultured in 2 mL medium on 3.5-cm dishes and in-duced into mature adipocyte. Cells were collected on day8 of differentiation and then lysed with precooled 800 mLof 80% methanol/water (v/v) for 20 min at 4�C. Half ofthe cell lysis solution was vacuum freeze dried and redis-solved in 200 mL of H2O. For measuring the a-KG in

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iWAT, up to 50 mg adipose tissue was weighted and ho-mogenized in 1 mL precooled 80% methanol/water (v/v).The supernatants were collected by centrifugation at12,000 rpm 4�C for 10 min. The samples were firstly sep-arated by a UPLC system using a Waters Acquity UPLCLC-20AB (ZIC-HILIC; Merck KGaA) (2.1 � 150 mm,5 mm) with a two-solvent system (A: 5 mL 1 mol/Lammonium acetate and 2 mL ammonia in 1 L double-dis-tilled H2O; and B: 100% acetonitrile) and a flow rate of0.2 mL/min and then analyzed by API 4000 Qtrap MassSpectrograph (AB Sciex API 4000 Qtrap; Thermo FisherScientific).

Ten-Eleven Translocation Hydroxylase Activity AssayC3H10T1/2 cells nuclei were isolated by using Nuclear andCytoplasmic Extraction Reagents (P0027; Beyotime Biotech-nology). Ten-eleven translocation hydroxylase (TET) activitywas determined by using the TET Hydroxylase Activity Quan-tification Kit (ab156913, Fluorometric; Abcam) according tothe manufacturer’s instructions.

Chromatin ImmunoprecipitationChromatin immunoprecipitation (ChIP) was performed aspreviously described (14). Briefly, C3H10T1/2 cells werefixed, lysed, and sonicated to obtain 200–1,000-bp DNAfragments. Soluble chromatin was precleared by using 30mL of Protein G Plus/Protein A-Agarose suspension (Cal-biochem) saturated with salmon sperm (1 mg/mL) andthen incubated with 1 mg anti-Prdm16 or PGC1a anti-body. Bound DNA–protein complexes were eluted, cross-links were reversed, and samples were phenol-chloroformextracted, ethanol precipitated, and used for QPCR withthe primers listed in Supplementary Table 2.

Hydroxymethylated and Methylated DNAImmunoprecipitationAccording to the method described previously (15), geno-mic DNA of C3H10T1/2 cells at day 8 of differentiationwas extracted by phenol-chloroform method. GenomicDNA (10 mg) was diluted in 300 mL TE buffer and soni-cated by 4�C Waterbath Sonicator for 12–16 cycles 30-son/30-s off, until the size of DNA was 300–1,000 bp. Thesonicated DNA was purified, and 10% of the sonicatedDNA was used as input sample. The sonicated DNA (3mg) was diluted in 450 mL TE buffer, 10 min at 95�C, andthen immediately cooled on ice along with adding 50 mL10 times IP buffer (100 mmol/L Na-phosphate, pH 7.0,1.4 mol/L NaCl, and 0.5% Triton X-100). Then, 5-hmc(A4001; Zymo Research) or 5-mc antibody (A3001; ZymoResearch) was added to the DNA solution with rotationovernight at 4�C. The DNA–antibody mixture was furtherincubated with preblocked Pierce magnetic protein A/G(88802; Thermo Scientific) for 2 h. The beads–antibody–DNAcomplex was washed three times with one time IP buffer andresponded in 250 mL digestion buffer (50 mmol/L Tris HCl,pH 8, 10 mmol/L EDTA, and 0.5% SDS) and then digested

with 7 mL proteinase K (10 mg/mL) by incubating at 50�Cfor 3 h. The purified DNA and input samples were quantizedby QPCR analysis using the primers listed in SupplementaryTable 2. Relative enrichment folds of indicated DNA regionswere determined after normalization to jnput, and 36b4 pro-moter was used as a negative control.

Statistical AnalysesGraphPad Prism 8 (GraphPad Software) was used for sta-tistical analysis. Only wounded animals were excludedfrom the analyses (n = 6 in this study). Data are pre-sented as mean ± SD. Comparisons between groups weremade using unpaired two-tailed Student t tests. For com-parison of three or more independent groups with onlyone variable, one-way analyses of variance plus Bonferronipost hoc tests were performed. For comparison of two ormore independent groups with two variables, two-wayANOVA plus Bonferroni post hoc tests were carried out.The statistical analyses were also indicated in the legendsof each figure, and P < 0.05 was considered significant.All experiments were repeated at least three times, andrepresentative data are shown.

Data and Resource AvailabilityThe data sets generated and/or analyzed during the cur-rent study are available from the corresponding authoron reasonable request. The resource generated duringand/or analyzed during the current study is availablefrom the corresponding author on reasonable request.

RESULTS

L-Theanine Promotes the Browning of SubcutaneousWAT in MiceThe metabolic effects of L-theanine were evaluated by i.p.administration of L-theanine into normal chow diet(NCD)–fed C57BL6 male mice. To examine the thermo-genic activity, the 8-week-old mice were treated with L-theanine or saline as a control once every day for 7 days.

After cold exposure (4�C) for 6 h, L-theanine–treated micehad a better capacity to maintain body temperature comparedwith controls (Fig. 1A). Mice treated with L-theanine showedless body weight gains as compared with saline-treated controlmice (Supplementary Fig. 1A). Adipose tissue oxygen con-sumption rate (OCR) is usually associated with mitochondrialenergy metabolism. L-Theanine significantly increased theOCR of WAT compared with the control group (Fig. 1B),which means enhanced mitochondrial function.

As is demonstrated in Fig. 1C, the abundance of mito-chondria in WAT was quantified by mtDNA copy number,which was increased by L-theanine treatment. Histologicalexamination of the subcutaneous (iWAT) and epididymalWAT (eWAT) revealed smaller adipocytes, which presentedbrown-like transformation of the WAT (Fig. 1D). We nextisolated RNA from the three different fat pads—eWAT,iWAT, and brown adipose tissue (BAT)—and comparedmRNA expression levels between L-theanine –treated mice

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and saline-treated mice. Expression levels of genes involvedin controlling energy expenditure and the thermogenic pro-gram, including Prdm16, PGC1a, Ucp1, mtTFA, Dio2, andCpt1b, were examined in iWAT, eWAT, and BAT. Althoughto different extents, L-theanine induced the expression ofthe above genes in iWAT (Fig. 1E), eWAT (SupplementaryFig. 1C), and BAT (Supplementary Fig. 1D), with its mostsignificant effect in iWAT (Fig. 1E). Besides Ucp1 and otherclassical thermogenic-related marker genes, such as Dio2and mtTFA, fatty acid oxidation–related gene expressionwas also significantly increased in iWAT (Fig. 1E). Impor-tantly, Western blot analysis shows that L-theanine couldobviously promote the protein expression of Prdm16,Ucp1, and PGC1a and the phosphorylation of AMPK iniWAT (Fig. 1F and Supplementary Fig. 1B). In BAT, Prdm16protein level and the phosphorylation of AMPK were slight-ly increased by L-theanine treatment (Supplementary Fig.

1E and F). Our data consistently showed that L-theaninecould promote the browning of WAT and thermogenic ca-pacity in mice.

L-Theanine Increases Energy Consumption in NCD-FedMiceAs the above results show, L-theanine promotes the brow-ning of WAT during cold exposure. With this in mind, wenext investigated the metabolic effect of L-theanine inmice using a comprehensive laboratory animal monitoringsystem. As shown in Fig. 2A–D, basal oxygen consump-tion and carbon dioxide release rates were significantlyhigher in mice administered L-theanine compared withcontrol subjects through a 12-h light/dark cycle. As shownin Fig. 2E, the L-theanine–treated mice showed a higherconsumption in the whole-body energy through a 12-hlight/dark cycle. The RER was calculated from VCO2 andVO2. Higher RER values suggest higher carbohydrate

Figure 1—L-Theanine treatment promotes subcutaneous fat brow-ning and adaptive thermogenesis in NCD-fed mice. The 8-week-oldC57BL6 NCD-fed male mice were administered L-theanine i.p. (100mg/kg/mouse, once a day) or saline as a control for 7 days. A: Rectaltemperature of the mice was recorded at the indicated time points af-ter cold exposure (4�C). B: Basal OCR of iWAT and eWAT tissues inmice after 6-h cold exposure was measured by Clark-type electrodeoxygraph and shown. C: Relative mtDNA copy number of iWAT andeWAT in mice after 6-h cold exposure. Data were normalized to thesaline group. D: Representative H&E staining images of BAT, eWAT,and iWAT of the mice after 6-h cold exposure. Scale bars, 40 mm. E:The mRNA expression of the indicated genes in iWAT after 6 h of coldexposure. n = 6. F: Immunoblotting for indicated proteins in iWAT after6-h cold exposure. For statistical analysis, two-way ANOVA and Bon-ferroni post hoc tests were performed in A, and unpaired two-tailedStudent t tests were performed in B, C, and E. All groups were com-pared with the saline group. All values are represented as means witherror bars representing SD. *P < 0.05, **P < 0.01, ***P < 0.001 ascompared with saline group. n = 6 for each group. ATP syn, ATP syn-thase; P-AMPK, phosphorylated AMPK.

Figure 2—L-Theanine increases energy expenditure in mice. Micewere treated as indicated in Fig. 1. Energy expenditure was evalu-ated by measurement of oxygen consumption (VO2) as shown in Aand B, carbon dioxide release (VCO2) as shown in C and D, andheat production of the mice as shown in E. The above parameterswere measured in mice placed in a metabolic cage during a 12:12-h light/dark cycle. Energy expenditure expressed as kcal/h per ani-mal. F: RER was calculated using equations described in ResearchDesign and Methods. The adjacent bar graphs represent the aver-age values for each group (B for A and D for C) (n = 6). Error barsrepresent SD, and significant differences compared with salinegroups are indicated by *P < 0.05, **P< 0.01 (Student t test).

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consumption, and lower RER values suggest more fat isused as the source of energy (16). As shown in Fig. 2F, L-theanine treatment decreased RER of the mice, demon-strating a tendency to fatty acid oxidation (17). Taken to-gether, these results indicate that L-theanine promotesthe whole-body energy expenditure and increase fattyacid combustion in NCD-fed mice.

L-Theanine Induces a Brown Adipocyte-like Feature inAdipocytes In VitroBecause the browning of subcutaneous WAT in vivo iscomplicated, we examined whether L-theanine induces

adipocyte browning directly. To better investigate the roleof L-theanine in the browning of adipocytes and its under-lying mechanism, we used the C3H10T1/2 cell line, whichis a mesenchymal stem cell line and can be induced to be-come mature adipocytes in vitro. We treated the adipo-cytes derived from C3H10T1/2 cells with L-theanine atdifferent concentrations of 0 mmol/L, 10 mmol/L, 50mmol/L, and 100 mmol/L. To evaluate the possible cyto-toxicity of L-theanine, we assessed LDH leakage after 24 hof exposure to L-theanine. Exposure of the adipocytes to10mmol/L, 50 mmol/L, and 100 mmol/L of L-theanine didnot influence the leakage rate of LDH (Fig. 3A), which

Figure 3—L-Theanine induces a brown adipocyte-like phenotype in the adipocytes derived from C3H10T1/2 cells in vitro. The adipocyteswere treated with L-theanine at the indicated dose or with saline as a control. After 48 h of treatment, cells were harvested for analyses. A:Relative LDH release from C3H10T1/2 exposed to saline or three different concentrations of L-theanine (10 mmol/L, 50 mmol/L, and 100mmol/L). B: Basal OCR of C3H10T1/2 was measured by Clark-type electrode oxygraph and shown. C: mtDNA copy number of C3H10T1/2. Data were normalized to the saline group. D: Cells were stained with MTG and Hoechst, and representative images are shown. Scalebar, 100 mm. Staining was performed after 48 h of treatment with saline or 50 mmol/L L-theanine. E: The mRNA levels of the indicatedgenes were determined by using RT-qPCR. Data were normalized to the saline group. F: Cell lysates were then subjected to Western blot-ting by using the indicated antibodies. Hsp90 serves as an internal control. G: Quantification of Western blotting was done by using Im-ageJ and shown. H: Oil Red O staining of differentiated adipocytes. Scale bar, 50 mm. For statistical analysis, two-way ANOVA andBonferroni post hoc tests were performed in A–C, and unpaired two-tailed Student t tests were performed in E and G. All groups werecompared with the saline group. All values are represented as means with error bars representing SD. *P < 0.05, **P < 0.01, ***P <0.001 as compared with saline group. n = 5 for each group. ATP syn, ATP synthase; P-AMPK, phosphorylated AMPK.

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suggests that L-theanine was not toxic to C3H10T1/2 atthe above three concentrations. We then measuredC3H10T1/2 OCR using a Clark-type electrode oxygraph(Fig. 3B). The OCR of 50 mmol/L and 100 mmol/L L-thea-nine–treated cells was significantly greater than that ofcontrols. Increase of mitochondrial contents was also ob-served in L-theanine–treated cells (Fig. 3C). MTG stainingshowed that L-theanine increased mitochondrial abun-dance (Fig. 3D), which was consistent with the results ofthe above mtDNA content analysis. L-Theanine treatmentof the adipocytes upregulated the mRNA levels of thegenes involved in the activation of energy expenditureand thermogenesis, including Prdm16, PGC1a, Ucp1,mtTFA, and Cpt1b (Fig. 3E) in the L-theanine dose-depen-dent manner. Western blot analysis further confirmedthat Prdm16 and Ucp1 protein were significantly upregu-lated by L-theanine at the concentrations of 50 mmol/Land 100 mmol/L (Fig. 3F and G). Similar effects of L-thea-nine were also observed in primary iWAT adipocytes(Supplementary Fig. 2). The lipid droplets in adipocyteswere stained by Oil Red O, and the result was shown inFig. 3H. The adipocytes derived from C3H10T1/2 cellstreated with L-theanine manifested diminishing tendencyof lipid droplet size, as indicated by Oil Red O staining,which is consistent with the greater energy expenditurecaused by L-theanine treatment (Fig. 3H). All of the abovephenotypes present enhanced acquisition of brown-like adi-pocyte features. These results demonstrate that L-theaninecould directly activate the browning of adipocytes in vitro.

Knockdown of Prdm16 Blunts the Role of L-Theaninein Promoting the Brown Adipocyte-like Features in theAdipocytes Derived From C3H10T1/2 CellsPrdm16 transcriptional complex is a dominant activatorof brown/beige adipocyte development (18). The above re-sults show that Prdm16 expression was obviously inducedby L-theanine treatment both in vivo and in vitro. Thebinding of Prdm16 to the enhancer and transcriptionstart site region of Ucp1 and Dio2 was significantly en-hanced by L-theanine treatment (Fig. 4A). PGC1a is a keyregulator of adipose tissue energy metabolism, especiallywhen involved in mitochondrial biosynthesis and thermo-genesis (19). As shown in Fig. 4B, L-theanine–induced bindingof PGC1a to the above regions is less obvious. Next, we con-structed adenovirus harboring shLacZ or shPrdm16 to infectthe adipocytes. The L-theanine–mediated upregulation of brow-ning gene expression was blunted upon the knockdown ofPrdm16 (Fig. 4C). Consistently, the induction of Ucp1 proteinexpression by L-theanine was abolished in shPrdm16-treatedcells (Fig. 4D and Supplementary Fig. 3). Oxygen electrode ex-periments and MTG staining showed that the effects of L-thea-nine treatment on promoting mitochondria respiration andincreasing mitochondrial abundance were eliminated by theknockdown of Prdm16 (Fig. 4E and F, respectively). These re-sults indicate that Prdm16 plays an essential role in L-theanine–mediated browning of adipocytes.

Adenovirus-Mediated Specific Knockdown of Prdm16 iniWAT Eliminates L-Theanine–Induced Browning of iWATPrdm16 promotes browning of iWAT by activating a brownfat-like gene program (20). Given the importance of Prdm16in L-theanine–mediated browning of C3H10T1/2 and in orderto clarify the role of Prdm16 in L-theanine effects on browningof WAT and energy expenditure in vivo, mice were subjectedto subcutaneous injection at the iWAT with adenovirus har-boring shLacZ or shPrdm16 and then were administered L-theanine or saline for 1 week (Fig. 5A). We isolated the SVFand mature adipocyte fraction from iWAT and performedRT-qPCR to examine the expression level of Prdm16. Asshown in Supplementary Fig. 4, the expression level ofPrdm16 in the mature adipocyte fraction was significantlydownregulated by shPrdm16. It should be noted that Prdm16expression level was also knocked down by Prdm16 in SVF,which indicates that the adenovirus can infect not only matureadipocytes, but also other cell types in iWAT. We measuredthe rectal temperature of mice every hour during the 6 h ofcold exposure. The results show that mice administeredshPrdm16 adenovirus at the iWAT displayed impaired coldtolerance compared with the mice injected with shLacZ adeno-virus (Fig. 5B). L-Theanine treatment significantly elevated rec-tal temperature of shLacZ-treated mice at cold temperatures,whereas it did not obviously impact that of shPrdm16-treatedmice. Moreover, L-theanine treatment increased the mRNAlevels of browning markers such as Prdm16, Ucp1, Cidea, andDio2 in iWAT of control mice, whereas it had no obviouseffect on the above genes’ expression in the iWAT ofshPrdm16-treated mice (Fig. 5C). Gene expression levels werealso examined in eWAT (Supplementary Fig. 5A). Knockdownof Prdm16 in iWAT did not affect the expression level ofPrdm16 in eWAT (shLacZ1Saline vs. shPrdm161Saline). Inthe control shRNA group (shLacZ group), treatment of themice with L-theanine increased the expression of browningmarker genes in eWAT, such as Prdm16, UCP1, Cidea, andCox8b (shLacZ1Saline vs. shLacZ1L-theanine). However,knockdown of Prdm16 in iWAT blunted the ability of L-thea-nine to promote the expression of browning marker genes ineWAT (shPrdm161Saline vs. shPrdm161L-theanine). Theremay be an L-theanine–triggered signal from iWAT to eWATthrough some endocrine factors to enhance the browninggene expression in eWAT, which could be impaired by theknockdown of Prdm16 in iWAT. Further studies are neededto investigate the above hypothesis. Meanwhile, L-theaninefailed to promote adipose tissue oxygen consumption in theiWAT of shPrdm16-treated mice (Fig. 5D). Similarly, thePrdm16 and Ucp1 protein expression levels were upregulatedupon L-theanine treatment in shLacZ-treated mice, whereasthey stayed unchanged in the iWAT of shPrdm16-treatedmice (Fig. 5E and Supplementary Fig. 5B). H&E staining ofthe inguinal WATs showed that L-theanine decreased adipo-cyte size and increased Ucp1 IHC-positive areas in shLacZ con-trol groups. However, these effects were abolished in shPrdm16groups (Fig. 5F). Therefore, we conclude that Prdm16 expres-sion is essential for the L-theanine–mediated browning effecton the iWAT and proadaptive thermogenesis in mice.

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Figure 4—Prdm16 plays an indispensable role in L-theanine–induced browning in the adipocytes derived from C3H10T1/2 cells. A and B:ChIP QPCR analysis for Prdm16 and PGC1a binding to the enhancer or promoter regions of the indicated genes in cultured adipocytes.The adipocytes were treated with saline or 50 mmol/L L-theanine for 48 h. Then, Prdm16 and PGC1a were processed by ChIP fromC3H10T1/2 and analyzed for their binding at the indicated regions. Data were normalized to saline group. Shown is a representative ChIPQPCR. C–F: C3H10T1/2 cells were infected with shLacZ or shPrdm16 adenoviruses as indicated, and medium was changed 48 h later.Then, the cells infected with adenovirus were treated with saline or 50 mmol/L L-theanine for 48 h. The following experiments were thenperformed. C: The mRNA levels of the indicated genes. D: Representative Western blot images of the indicated proteins. E: OCR in cells.F: Cells were stained with MTG and Hoechst, and representative images are shown. Scale bar, 100 mm. Data are expressed as mean ±SD. *P < 0.05, **P < 0.01 as compared with saline group in A and B or compared with shLacZ1saline group in C and E by Student t test.TSS, transcription start site.

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AMPK Activation Is Indispensable forL-Theanine–Induced Browning of Adipocytes

AMPK is a crucial cellular energy sensor that has recentlybeen demonstrated to be important in regulating the meta-bolic activity of brown-like adipocytes (21). It was found thatthe phosphorylation of AMPK was activated by L-theanine in

mice livers (22). We therefore investigated the role of L-thea-nine in the activation of AMPK in adipocytes. Our resultsdemonstrated that L-theanine induced the phosphorylationof AMPK both in the adipocytes (Fig. 3F) and in iWAT (Fig.1F) of the mice. Therefore, we sought to explore if L-theaninepromotes the browning of adipocytes via activating the

Figure 5—Prdm16 plays an indispensable role in L-theanine–induced iWAT browning in mice. Eight-week-old male WT mice were injectedsubcutaneously (i.h.) into iWAT with adenovirus of shLacZ or shPrdm16 twice a week for 2 weeks. The mice were i.p. injected with salineor L-theanine in the 2nd week for 7 days (d). Then, the following experiments were performed. A: Schematic description of animal experi-ments. B: Rectal temperature of the mice was recorded at the indicated time points after cold exposure (4�C). C: The mRNA levels of theindicated genes. n = 6. D: OCR of iWAT was measured by Clark-type electrode oxygraph and shown. E: Western blot analysis of the pro-tein levels in iWAT by using the indicated antibodies. Hsp90 serves as an internal control. F: H&E staining (HE) and IHC for UCP1 protein iniWAT sections of the mice. Scale bars, 50 mm. n = 6. Data are expressed as mean ± SD. *P< 0.05, **P< 0.01 compared with shLacZ1sa-line group by Student t test.

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AMPK signaling pathway. The shRNA-mediated knockdownof the AMPKa subunit was performed in the adipocytes. Asshown in Fig. 6A, knocking down of AMPK blunted the roleof L-theanine in inducing the mRNA expression of Prdm16and Ucp1. Western blotting showed that knockdown ofAMPK reduced the expression of Ucp1 and Prdm16 promot-ed by L-theanine (Fig. 6B and C and Supplementary Fig. 6).As shown in Fig. 6D, L-theanine promoted a significant in-crease in oxygen consumption in C3H10T1/2, and this in-crease was completely abrogated by AMPK knockdown inC3H10T1/2. Enhanced mitochondrial abundance by L-thea-nine was also abolished in C3H10T1/2 infected withshAMPK adenovirus (Fig. 6E). As downregulation of AMPKsignaling blunted the role of L-theanine in inducing the

expression of Prdm16, it is suggested that AMPK andPrdm16 were both involved in L-theanine–mediated brow-ning of adipocytes, and Prdm16 could be a downstream tar-get of AMPK. These data demonstrate a potentialL-theanine/AMPK/Prdm16 pathway activation in L-thea-nine–mediated browning of adipocytes.

L-Theanine Increases the Cellular Content ofTricarboxylic Acid Metabolite a-KG and Decreasesthe DNA Methylation Level of Prdm16 Promoter inC3H10T1/2As Prdm16 plays an important role in L-theanine–mediatedbrowning of adipocytes, the mechanism of how Prdm16 ex-pression is regulated was then investigated. DNA methylation

Figure 6—AMPK activation is necessary for L-theanine to promote adipocyte browning. C3H10T1/2 cells were infected with shLacZ or shAMPKaadenoviruses as indicated, andmediumwas changed 48 h later. Then, the cells infected with adenovirus were treated with saline or 50 mmol/L L-the-anine for 48 h. The following experiments were then performed. A: The mRNA levels of the indicated genes. B: Cell lysates were then subjected toWestern blotting by using the indicated antibodies. Hsp90 serves as an internal control. C: Statistical analysis of Western blotting gray-level resultsfor B. D: OCR in cells was measured. E: Cells were stained with MTG and Hoechst, and representative images are shown. Scale bar, 100 mm. Dataare expressed as mean ± SD. *P< 0.05, **P< 0.01 compared with shLacZ1saline group by Student t test.

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is a complicated process and plays essential roles in regulatinggene expression. Based on a methylated and hydroxymethy-lated DNA immunoprecipitation procedure, we further studiedDNA methylation (5-mc) and hydroxymethylation (5-hmc) inthe Prdm16 promoter in three different regions of Prdm16promoter. As shown in Fig. 7A and B, L-theanine significantlydecreased 5-mc level and increased 5-hmc level in Prdm16 pro-moter. The tricarboxylic acid cycle (TCA) metabolite a-KG is

known to facilitate DNA demethylation. The a-KG concentra-tion was determined by liquid chromatography-tandem massspectrometry and showed a significant elevation in L-theanine–-treated adipocytes (Fig. 7C). Moreover, the content of a-KGwas increased in the iWAT of L-theanine–treated mice (Fig. 7D).

Given that the TETs, including TET1, 2, and 3, catalyzeactive DNA demethylation, the effect of L-theanine onTET activity was examined. It was found that L-theanine

Figure 7—L-Theanine increases a-KG in adipocytes and epigenetically decreases the DNA methylation of Prdm16 promoter. A: The adipocyteswere treated with saline or 50 mmol/L L-theanine for 48 h. Then, a methylated and hydroxymethylated DNA immunoprecipitation procedure was per-formed according to protocol. The a, b, and c primers were used to determine relative enrichment folds of three different regions in the Prdm16 pro-moter after normalization to inputs, and a 36b4 promoter region was used as a negative control. The enrichment level of 5-mc in Prdm16 promoteris shown. B: Cells were treated as in A, and the enrichment level of 5-hmc in Prdm16 promoter is shown. n = 3. C: Cells were treated as in A, andthe intracellular a-KG level was determined. D: Mice were treated as in Fig. 1, and the a-KG level in iWAT was measured. E: Cells were treated as inA, and the activity of TETs wasmeasured. F: Cells were treated as in Fig. 6, and the intracellular a-KG level was determined. n = 5. Cells were treatedas in F, and the enrichment level of 5-mc (G) and 5-hmc (H) on the Prdm16 promoter (using primers a as described in A) was examined. n = 3. Dataare expressed as mean ± SD. *P < 0.05, **P < 0.01, ***P< 0.001 compared with saline group or shLacZ1saline group by Student t test or one-way ANOVA followed by Dunnett multiple comparisons.

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significantly enhanced TET activity in the adipocytes de-rived from C3H10T1/2 cells (Fig. 7E). This result suggeststhat L-theanine–induced DNA demethylation on thePrdm16 promoter may be mediated by enhanced TET ac-tivity. Further, we found that L-theanine–mediated eleva-tion of a-KG concentration was significantly blunted inshAMPK-treated cells compared with the correspondingcontrol group (Fig. 7F). In addition, knocking down ofAMPK abolished the role of L-theanine in decreasing 5-mclevel and increasing 5-hmc level on the Prdm16 promoter(Fig. 7G and H). This suggests that AMPK is an essentialfactor in a-KG/Prdm16 axis and is an upstream regulatorof a-KG. AMPK signaling–mediated elevation of a-KGpromotes Prdm16 promoter DNA demethylation, therebystimulating Prdm16 expression. Overall, our data suggestthat L-theanine inhibits the DNA methylation of thePrdm16 promoter and facilitates Prdm16 expressionthrough the AMPK–a-KG pathway.

Antiobesity Effects of L-Theanine in HFD-InducedObese MiceBecause L-theanine promotes browning of adipose tissue,we aimed to investigate the potential antiobesity effectsof L-theanine. C57BL6 male mice were fed with HFD for16 weeks. From the 7th week of HFD feeding, mice wereinjected i.p. with saline or L-theanine (100 mg/kg), once aday for the next 10 consecutive weeks (Fig. 8A). L-Thea-nine did not affect the amount of food intake in mice(Supplementary Fig. 7A). As shown in Fig. 8B, L-theaninereduced the weight gain of mice induced by HFD, whichis associated with the reduced fat mass content (Fig. 8C).In addition, we weighed three types adipose tissue. L-The-anine significantly reduced tissue weight in iWAT andeWAT, while brown fat content did not statistically differbetween the two groups (Fig. 8D). Obesity is closely asso-ciated with elevation of lipids in plasma. The mice plasmawas collected and analyzed for TG fatty acid, TC, andFFA. Plasma TG, TC, and FFA levels were reduced in micetreated with L-theanine compared with saline-treatedmice (Fig. 8E). HFD-induced obesity is also closely relatedto glucose intolerance and insulin resistance. We nextevaluated whether L-theanine treatment ameliorates glu-cose intolerance and insulin resistance by conducting i.p.GTT and ITT (IP-GTT and IP-ITT, respectively). L-Thea-nine–treated mice exhibited an improved metabolic profilein glucose tolerance and insulin sensitivity (Fig. 8F–I). Weisolated the iWAT from these mice. Proteins and mRNAswere extracted from tissues in the two groups for QPCR andWestern blotting analysis. QPCR revealed significantlyincreased Prdm16, Ucp1, and other browning-related geneexpression in the L-theanine–treated mice (Fig. 8J). Westernblotting analysis demonstrated that the expression ofPrdm16 and Ucp1 were significantly elevated in the L-thea-nine–treated group compared with the saline control group(Fig. 8K and Supplementary Fig. 7B).

There was also an upregulation of thermogenic genesin eWAT, as shown in Supplementary Fig. 7C. There wasno expression level difference in most of the thermogenicgenes except Dio2 in BAT (Supplementary Fig. 7D). H&Estaining revealed that HFD-fed mice treated by L-theaninehad smaller adipocytes in iWAT, and IHC staining ofiWAT showed higher expression of Ucp1 (Fig. 8L). Asshown in Supplementary Fig. 7E, visceral adipocyte sizeswere reduced in mice administered L-theanine, and theadipocyte sizes in BAT were also slightly reduced. Further-more, metabolic cage studies and an OCR test of iWATwere performed at the 9th week (9 weeks of HFD feedingwith L-theanine treatment in the last 3 weeks). As shownin Supplementary Fig. 7F, 3 weeks of L-theanine treat-ment enhanced the OCR of iWAT in HFD-fed mice. In ad-dition, metabolic cage studies indicated that 3 weeks ofL-theanine treatment significantly promoted the basal ox-ygen consumption (Supplementary Fig. 7G) and energyexpenditure (Supplementary Fig.7H) of the whole bodyand mildly decreased RER (Supplementary Fig. 7I). Thesedata suggest that L-theanine–mediated resistance to die-t-induced obesity is driven by increased energy expendi-ture and increased iWAT thermogenesis. Overall, ourresults demonstrate that L-theanine activates the brow-ning of WAT and contributes to ameliorating diet-inducedobesity and related disorders in mice.

DISCUSSION

L-Theanine is a nonprotein amino acid mainly found intea plants. L-Theanine is an important taste- and health-related component in tea and is used as an indicator toevaluate tea functions (23). L-Theanine has multifacetedhealth benefits (24) and is considered as an index of teaquality (25). However, the role and mechanism of L-thea-nine in treating obesity and metabolic diseases is stillpoorly investigated. In this study, we proposed that L-the-anine promotes the browning of WAT, which contributesto the amelioration of obesity, the improvement of insu-lin sensitivity, and the reduction of plasma lipids in HFD-fed mice.

In this article, we found that NCD-fed mice i.p. admin-istered L-theanine showed an enhanced cold tolerance ca-pacity compared with control mice. Our study furtherdemonstrates that L-theanine activates the thermogenicprogram of adipose tissue in NCD-fed mice, with themost significant effects on iWAT (Fig. 1). L-Theanine sig-nificantly increases energy consumption in NCD-fed micecompared with the control group (Fig. 2). Moreover, usingprimary iWAT adipocytes and the adipocytes derived fromC3H10T1/2 cells as an in vitro cell culture model, wefound that L-theanine directly induced the browning ofadipocytes in vitro (Fig. 3 and Supplementary Fig. 2).Prdm16 was a critical transcriptional regulator (26) andan essential modulator in the downstream of L-theaninebecause knockdown of Prdm16 in the adipocytes bluntedthe role of L-theanine in the induction of a brown-like

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program in adipocytes (Fig. 4). Similarly, knockdown ofPrdm16 in iWAT inhibited L-theanine–mediated brown-likefat formation in mice (Fig. 5). AMPKa phosphorylationplayed an important role in L-theanine–induced browningof adipocytes. We demonstrated that the L-theanine–in-duced browning of adipocytes, including elevated Prdm16expression, was blocked by AMPKa knockdown (Fig. 6).We show a potential L-theanine/AMPK/Prdm16 pathwayactivation in L-theanine–induced browning of adipocytes(Fig. 7). We found that L-theanine significantly reduced the5-mc level and increased the 5-hmC level in the Prdm16

promoter. Furthermore, L-theanine increased the concen-tration of C3H10T1/2 intracellular a-KG, which wasblocked by the knockdown of AMPKa. a-KG is known tofacilitate DNA demethylation. Our results suggest thatL-theanine activates AMPK signaling to elevate a-KG con-centration in adipocytes, which leads to DNA demethyla-tion and decreases the DNA methylation level on thePrdm16 promoter, thereby facilitating Prdm16 expressionand ultimately promoting adipocyte browning (Fig. 7). Fur-thermore, HFD-fed mice administered L-theanine exhibitedimproved glucose and insulin sensitivity and reduced

Figure 8—L-Theanine reduces obesity in diet-induced obese mice. A: Schematic description of animal experiments. C57BL6 male micewere fed with HFD for 16 weeks. From the 7th week of HFD feeding, mice were injected i.p. with saline or L-theanine (100 mg/kg) once aday for the next 10 consecutive weeks. B: Body weight of mice after treatment of saline or L-theanine. C: Body composition of the mice af-ter 10 weeks of L-theanine or saline treatment. D: Tissue weight (normalized to body weight) of iWAT, eWAT, and BAT after 10 weeks of sa-line or L-theanine treatment. E: Plasma TG, TC, and FFA. F: Glucose concentrations during an IP-GTT. G: Area under the curve analysis ofIP-GTT. H: Glucose concentrations during an IP-ITT. I: Area under the curve analysis of IP-ITT. J: The mRNA levels of the indicated genesin iWAT. K: Tissue lysates were subjected to Western blotting by using the indicated antibodies. Hsp90 serves as an internal control. L:H&E (HE) and Ucp1 IHC staining of iWAT. Scale bars, 40 mm. For statistical analysis in B–J, data were compared between the saline groupand the L-theanine group. All values are represented as means with error bars representing SD. *P < 0.05; **P < 0.01; ***P < 0.001. n = 6for each group. 6w, 6 weeks; ATP syn, ATP synthase; P-AMPK, phosphorylated AMPK.

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plasma TG, TC, and FFA levels compared with saline--treated mice. (Fig. 8).

Browning of adipose tissue was inducible by exercise,cold exposure, and fasting in WAT, which has substantialmetabolic benefits (27). Compared with classical brownadipocytes, beige adipocytes have a better inducibility to-ward thermogenic gene expression, especially Ucp1, andtherefore a higher thermogenic capacity in response tostimulus (28). Accordingly, thermogenesis, which meansdispersing energy in the form of heat, will contribute tomaintaining metabolic homeostasis through enhancingenergy expenditure. This process can be achieved by theactivation of Ucp1, an uncoupling hallmark mitochondriainner membrane protein abundantly expressed in BAT.Ucp1 induces uncoupling respiration and mitochondrialATP synthesis by reducing the proton gradient across themitochondrial inner membrane (29). Ucp1 ablation is re-ported to induce obesity and abolish thermogenesis atthermoneutrality (30). Our data suggest a functional roleof L-theanine in inducing the browning of white adipo-cytes, which help to improve thermogenic capacity andcombat obesity in mice.

AMPK plays an important role in energy metabolismand mitochondrial biogenesis (31,32). However, themechanisms by which AMPK regulates metabolism arenot completely understood. Many studies have shownthat activating AMPK can improve metabolic rate andhelp to resist obesity. For instance, metformin promotesbrowning of adipose tissue and prevents fatty liver (33).Taurine activates AMPK to promote browning of WATand combat obesity in mice (34). Obesity and glucosemetabolic disorder are closely associated with a decreasein AMPK activity (35). Our results identified that themaster regulator AMPK is involved in the L-theanine–me-diated WAT thermogenic program.

The structure of L-theanine is similar to that of L-gluta-mine. L-Glutamine is conditionally essential and the mostabundant amino acid in the body. It acts as an importantenergy source and supplies carbons to the TCA cycle toproduce ATP. Therefore, insufficiency of L-glutaminecould cause an energy shortage and lead to AMPK activa-tion (36). It has been reported that L-theanine could com-pete with L-glutamine in binding to the L-glutaminetransporter on the cell surface, which is responsible forthe incorporation of extracellular L-glutamine (37). Inthis way, L-theanine could inhibit the uptake of L-gluta-mine by the cells. Therefore, L-theanine treatment of theadipocytes may inhibit the uptake of L-glutamine and de-crease the intracellular L-glutamine level, which wouldlead to an energy shortage and activate AMPK phosphory-lation. Further studies are needed to investigate the abovehypothesis.

Our data revealed a functional role of L-theanine in theactivation of thermogenic program in adipocytes, whichis shown to be involved in the increased energy expendi-ture and antiobesity function. Indeed, our results show

that L-theanine could activate differentiated C3H10T1/2and iWAT SVF adipocytes to express more thermogenic-related genes and exhibit brown adipocyte-like pheno-types. We demonstrated that transdifferentiation ofadipocytes could be induced by L-theanine in vitro. L-The-anine could upregulate the expression of many browning-associated genes. The most prominent shift in this pro-cess was Prdm16. Notably, Prdm16 is a critical regulatorof browning and the thermogenic gene expression pro-gram (20,38). Raising the Prdm16 level ameliorates fibro-sis of aged mice and promotes beige adipocyte activation(39). Thus, Prdm16 plays an important role in L-theani-ne–mediated browning of white adipocytes.

C3H10T1/2 and iWAT exhibit a significant increase ina-KG concentration after L-theanine treatment, whichcould facilitate DNA demethylation of Prdm16. Beneficialeffects of a-KG on extending the life span of adult wormshave been reported (40). a-KG is a product of the TCA cy-cle and produced from isocitrate by isocitrate dehydroge-nase, which can also be produced from glutamate byglutamate dehydrogenase. It functions as a cofactor ofTETs to catalyze DNA demethylation. Inhibition of theAMPK/a-KG signaling pathway suppressed BAT develop-ment, which contributes to obesity (41). Our results re-veal an L-theanine/AMPK/a-KG/Prdm16 axis that inducesthe brown-like features in white adipocytes.

In conclusion, our results show a previously unknownrole for L-theanine in regulating adipose tissue and adipo-cyte metabolism. We identified that L-theanine promotesthe browning of adipose tissue in NCD- and HFD-fedmice, which ameliorates obesity, improves glucose and in-sulin tolerance, and reduces levels of plasma lipids(Supplementary Fig. 8). Thus, L-theanine may provide anapproach to help combat obesity and obesity-associateddisorders based on its positive role in WAT browning.

Funding. This study was supported by the National Key R&D Program ofChina (2018YFA0800401 to Q.-Q.T.) and National Natural Science Foundationof China grants (32070751 and 31871435 to L.G. and 81730021 and32070760 to Q.-Q.T.).Duality of Interest. No potential conflicts of interest relevant to thisarticle were reported.Author Contributions. W.-Q.P. and L.G. were involved in study de-sign, conducted the experiments, analyzed the data, and drafted the paper.G.X., B.-Y.L., and Y.-Y.G. performed the experiments. L.G. and Q.-Q.T. de-signed and supervised the study and wrote the paper. W.-Q.P. and L.G. arethe guarantors of this work and, as such, had full access to all of the data inthe study and take responsibility for the integrity of the data and the accura-cy of the data analysis.

References

1. Cohen P, Levy JD, Zhang Y, et al. Ablation of PRDM16 and beige adiposecauses metabolic dysfunction and a subcutaneous to visceral fat switch. Cell2014;156:304–3162. Shan T, Xiong Y, Zhang P, et al. Lkb1 controls brown adipose tissue

growth and thermogenesis by regulating the intracellular localization ofCRTC3. Nat Commun 2016;7:12205

diabetes.diabetesjournals.org Peng and Associates 1471

3. Pellegrinelli V, Carobbio S, Vidal-Puig A. Adipose tissue plasticity: howfat depots respond differently to pathophysiological cues. Diabetologia2016;59:1075–10884. Chouchani ET, Kazak L, Spiegelman BM. New advances in adaptivethermogenesis: UCP1 and beyond. Cell Metab 2019;29:27–375. Sae-Tan S, Rogers CJ, Lambert JD. Decaffeinated green tea andvoluntary exercise induce gene changes related to beige adipocyte formationin high fat-fed obese mice. J Funct Foods 2015;14:210–2146. Zhou TS, Zhou R, Yu YB, et al. Cloning and characterization of a flavonoid30-hydroxylase gene from tea plant (Camellia sinensis). Int J Mol Sci2016;17:2617. Ekborg-Ott KH, Taylor A, Armstrong DW. Varietal differences in the totaland enantiomeric composition of theanine in tea. J Agric Food Chem1997;45:353–3638. Vuong QV, Bowyer MC, Roach PD. L-Theanine: properties, synthesis andisolation from tea. J Sci Food Agric 2011;91:1931–19399. Soukas A, Socci ND, Saatkamp BD, Novelli S, Friedman JM. Distincttranscriptional profiles of adipogenesis in vivo and in vitro. J Biol Chem2001;276:34167–3417410. Guo L, Zhang P, Chen Z, et al. Hepatic neuregulin 4 signaling defines anendocrine checkpoint for steatosis-to-NASH progression. J Clin Invest2017;127:4449–446111. Livak KJ, Schmittgen TD. Analysis of relative gene expression data usingreal-time quantitative PCR and the 2(-Delta Delta C(T)) Method. Methods2001;25:402–40812. Lv LH, Yu JD, Li GL, et al. Functional distinction of rat liver natural killercells from spleen natural killer cells under normal and acidic conditionsin vitro. Hepatobiliary Pancreat Dis Int 2012;11:285–29313. Park SJ, Gavrilova O, Brown AL, et al. DNA-PK promotes themitochondrial, metabolic, and physical decline that occurs during aging. CellMetab 2017;26:44714. Kurtenbach S, Reddy R, Harbour JW. ChIPprimersDB: a public repositoryof verified qPCR primers for chromatin immunoprecipitation (ChIP). NucleicAcids Res 2019;47(D1):D46–D4915. Mohn F, Weber M, Sch€ubeler D, Roloff TC. Methylated DNAimmunoprecipitation (MeDIP). Methods Mol Biol 2009;507:55–6416. Arao Y, Hamilton KJ, Lierz SL, Korach KS. N-terminal transactivationfunction, AF-1, of estrogen receptor alpha controls obesity throughenhancement of energy expenditure. Mol Metab 2018;18:68–7817. Lu P, Zhang FC, Qian SW, et al. Artemisinin derivatives prevent obesityby inducing browning of WAT and enhancing BAT function. Cell Res2016;26:1169–117218. Hasegawa Y, Ikeda K, Chen Y, et al. Repression of adipose tissue fibrosisthrough a PRDM16-GTF2IRD1 complex improves systemic glucose homeostasis. CellMetab 2018;27:180–194.e619. Zhang J, Wu H, Ma S, et al. Transcription regulators and hormonesinvolved in the development of brown fat and white fat browning:transcriptional and hormonal control of brown/beige fat development. PhysiolRes 2018;67:347–36220. Seale P, Conroe HM, Estall J, et al. Prdm16 determines the thermogenic programof subcutaneous white adipose tissue in mice. J Clin Invest 2011;121:96–10521. Desjardins EM, Steinberg GR. Emerging role of AMPK in brown and beigeadipose tissue (BAT): implications for obesity, insulin resistance, and type 2diabetes. Curr Diab Rep 2018;18:80

22. Lin L, Zeng L, Liu A, et al. l-Theanine regulates glucose, lipid, andprotein metabolism via insulin and AMP-activated protein kinase signalingpathways. Food Funct 2020;11:1798–180923. Li JL, Zhou Y, Zhou B, et al. Habitat management as a safe and effectiveapproach for improving yield and quality of tea (Camellia sinensis) leaves. SciRep 2019;9:43324. T€urk€oz€u D, Sanlier N. L-theanine, unique amino acid of tea, and itsmetabolism, health effects, and safety. Crit Rev Food Sci Nutr 2017;57:1681–168725. Li X, Zhang L, Ahammed GJ, et al. Stimulation in primary and secondarymetabolism by elevated carbon dioxide alters green tea quality in Camelliasinensis L. Sci Rep 2017;7:793726. Kissig M, Ishibashi J, Harms MJ, et al. PRDM16 represses the type Iinterferon response in adipocytes to promote mitochondrial and thermogenicprograming. EMBO J 2017;36:1528–154227. Bl�eher M, Meshko B, Cacciapuoti I, et al. Egr1 loss-of-function promotesbeige adipocyte differentiation and activation specifically in inguinalsubcutaneous white adipose tissue. Sci Rep 2020;10:1584228. Vegiopoulos A, M€uller-Decker K, Strzoda D, et al. Cyclooxygenase-2controls energy homeostasis in mice by de novo recruitment of brownadipocytes. Science 2010;328:1158–116129. Kajimura S. Promoting brown and beige adipocyte biogenesis through thePRDM16 pathway. Int J Obes Suppl 2015;5(Suppl. 1):S11–S1430. Feldmann HM, Golozoubova V, Cannon B, Nedergaard J. UCP1 ablationinduces obesity and abolishes diet-induced thermogenesis in mice exemptfrom thermal stress by living at thermoneutrality. Cell Metab2009;9:203–20931. Zong H, Ren JM, Young LH, et al. AMP kinase is required formitochondrial biogenesis in skeletal muscle in response to chronic energydeprivation. Proc Natl Acad Sci USA 2002;99:15983–1598732. Cant�o C, Gerhart-Hines Z, Feige JN, et al. AMPK regulates energyexpenditure by modulating NAD1 metabolism and SIRT1 activity. Nature2009;458:1056–106033. Kim EK, Lee SH, Jhun JY, et al. Metformin prevents fatty liver andimproves balance of white/brown adipose in an obesity mouse model byinducing FGF21. Mediators Inflamm 2016;2016:581303034. Guo YY, Li BY, Peng WQ, Guo L, Tang QQ. Taurine-mediated browning ofwhite adipose tissue is involved in its anti-obesity effect in mice. J Biol Chem2019;294:15014–1502435. Ruderman NB, Carling D, Prentki M, Cacicedo JM. AMPK, insulinresistance, and the metabolic syndrome. J Clin Invest 2013;123:2764–277236. Lie S, Wang TT, Forbes B, Proud CG, Petersen J. The ability to utiliseammonia as nitrogen source is cell type specific and intricately linked to GDH,AMPK and mTORC1. Sci Rep 2019;9:146137. Yoneda Y. An L-glutamine transporter isoform for neurogenesis facilitatedby L-theanine. Neurochem Res 2017;42:2686–269738. Seale P, Kajimura S, Yang W, et al. Transcriptional control of brown fatdetermination by PRDM16. Cell Metab 2007;6:38–5439. Wang W, Ishibashi J, Trefely S, et al. A PRDM16-driven metabolic signalfrom adipocytes regulates precursor cell fate. Cell Metab 2019;30:174–189.e540. Chin RM, Fu X, Pai MY, et al. The metabolite a-ketoglutarate extendslifespan by inhibiting ATP synthase and TOR. Nature 2014;510:397–40141. Yang Q, Liang X, Sun X, et al. AMPK/a-ketoglutarate axis dynamicallymediates DNA demethylation in the Prdm16 promoter and brown adipogenesis. CellMetab 2016;24:542–554

1472 L-Theanine Activates WAT Browning Diabetes Volume 70, July 2021